NLS peptide conjugated molecular beacons for visualizing nuclear RNA in living cells.
Imaging the expression and localization of RNAs in live-cell nucleus can provide important information on RNA synthesis, processing, and transport. Here, we report the development of a bifunctional molecular beacon (NLS-MB) composed of a single nuclear localization sequence (NLS) peptide conjugated to a molecular beacon for efficient delivery and imaging of endogenous RNAs in the nuclei of living cells. We characterized the NLS-MBs by comparing their signal-to-noise ratios with unmodified molecular beacons and determined their efficiency of nuclear import. We demonstrated the specificity and sensitivity of the method by observing in living cells the localization and colocalization of small nuclear RNAs (snRNA) U1 and U2 at discrete foci in the nucleoplasm, and the localization of small nucleolar RNA U3 in the nucleolus. These snRNAs were chosen because of their essential roles in RNA biogenesis. The results were validated using in situ hybridization as positive control and random beacons as negative control. This novel approach may be applied to imaging other nuclear RNAs and pre-mRNAs in living cells.
Nitin
Nitin
N
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.
Bao
Gang
G
D001483:Base Sequence; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000378:metabolism; D015195:Drug Design; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019913:Nuclear Localization Signals / Q000138:chemical synthesis / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012342:RNA, Small Nuclear / Q000032:analysis* / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000032:analysis / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D019913:Nuclear Localization Signals; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar